Kinetic investigation of rat liver microsomal electron transport from NADH to cytochrome P-450.
NADH-dependent reduction of microsomal cytochrome P-450 has been analyzed kinetically by observing formation of the ferrous-carbonyl complex. Reduction is best described by two exponential equations with apparent first-order rate constants of 1.49 +/- 0.20 and 0.077 +/- 0.015 min-1. By either selective removal or inhibition of specific electron carriers, the kinetic data of minimally altered microsomes are further resolved into a third slow phase. Either addition of anti-cytochrome b5 immune globulin or reduction of cytochrome b5 with ascorbic acid markedly diminishes only the third phase. In reconstitution of purified flavoproteins, phospholipids, and a single isozymic form of cytochrome P-450, without cytochrome b5 only biphasic reduction of cytochrome P-450 is observed. Thus, microsomal cytochrome P-450 appears to be reduced via two independent pathways of electron transport from NADH; the biphasic reduction occurs via cytochrome P-450 reductase while the slower monophasic reduction occurs via cytochrome b5. Multiphasic reduction occurs via cytochrome b5. Multiphasic kinetics are not altered by in vivo inductions of different isozymes of cytochrome P-450. Accordingly, the rates of reduction appear to be an intrinsic property of the electron transport process and not directed by the heterogeneity of the isozymic mixture of ultimate electron acceptors.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003580:Cytochromes / Q000378:metabolism; D015786:Cytochromes b5; D004579:Electron Transport; D007700:Kinetics; D008634:Mersalyl / Q000494:pharmacology; D008862:Microsomes, Liver / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats
D003580:Cytochromes; D009243:NAD; D008634:Mersalyl; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D009245:NADH Dehydrogenase